Product Name:	Mouse Transcription factor GATA-4 (Gata4) ELISA Kit
SKU:	MOEB0323
Size:	96T
Target:	Mouse Transcription factor GATA-4 (Gata4)
Synonyms:	GATA-binding factor 4, Gata-4
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	0.156-10ng/mL
Sensitivity:	0.09ng/mL

Intra CV:	Provided with the Kit
Inter CV:	Provided with the Kit
Linearity:	Provided with the Kit
Recovery:	Provided with the Kit
Function:	Transcriptional activator that binds to the consensus sequence 5'-AGATAG-3' and plays a key role in cardiac development (By similarity). In cooperation with TBX5, it binds to cardiac super-enhancers and promotes cardiomyocyte gene expression, while it downregulates endocardial and endothelial gene expression (By similarity). Involved in bone morphogenetic protein (BMP)-mediated induction of cardiac-specific gene expression (By similarity). Binds to BMP response element (BMPRE) DNA sequences within cardiac activating regions (By similarity). Acts as a transcriptional activator of ANF in cooperation with NKX2-5 (PubMed:9584153). Promotes cardiac myocyte enlargement (By similarity). Required during testicular development (By similarity). May play a role in sphingolipid signaling by regulating the expression of sphingosine-1-phosphate degrading enzyme, spingosine-1-phosphate lyase.

Uniprot:	Q08369
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse Transcription factor GATA-4
Sub Unit:	Interacts with ZNF260 (By similarity). Interacts with the homeobox domain of NKX2-5 through its C-terminal zinc finger. Also interacts with JARID2 which represses its ability to activate transcription of ANF. Interacts with NFATC4 and LMCD1. Forms a complex made of CDK9, CCNT1/cyclin-T1, EP300 and GATA4 that stimulates hypertrophy in cardiomyocytes. Interacts with NR5A1, ZFPM2 and TBX5. Interacts with TBX18.
Subcellular Location:	Nucleus
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	GATA4: a conserved and ubiquitous member of the GATA family of zinc-finger transcription factors. Members of this family recognize the GATA motif which is present in the promoters of many genes. This protein is thought to regulate genes involved in embryogenesis and in myocardial differentiation and function. May regulate a set of cardiac-specific genes and play a crucial role in cardiogenesis. Defects are a cause of atrial septal defect 2 (ASD2). ASD2 is an autosomal dominant condition with atrial septal defect and other congenital heart disease but no conduction defects or noncardiac abnormalities.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Transcription factor Cellular Component: nuclear chromatin; nucleus; transcription factor complex Molecular Function:chromatin binding; DNA binding; metal ion binding; NFAT protein binding; protein binding; protein kinase binding; RNA polymerase II transcription factor activity, enhancer binding; sequence-specific DNA binding; transcription activator binding; transcription coactivator activity; transcription factor activity; transcription factor binding; zinc ion binding Biological Process: BMP signaling pathway; cardiac muscle cell differentiation; cardiac muscle development; cardiac muscle hypertrophy; cell development; cell-cell signaling; embryonic digestive tract morphogenesis; embryonic foregut morphogenesis; embryonic heart tube anterior/posterior pattern formation; embryonic heart tube development; embryonic morphogenesis; endoderm formation; gastrulation with mouth forming second; heart development; heart looping; heart morphogenesis; in utero embryonic development; male gonad development; negative regulation of autophagy; positive regulation of angiogenesis; positive regulation of BMP signaling pathway; positive regulation of cardiac muscle cell proliferation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of cardiac muscle cell proliferation; regulation of gene expression; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-dependent; response to drug; response to estrogen stimulus; response to mechanical stimulus; response to retinoic acid; Sertoli cell differentiation; spermatogenesis; transcription from RNA polymerase II promoter; transcription, DNA-dependent; tube morphogenesis; Wnt receptor signaling pathway through beta-catenin
UniProt Code:	Q08369
NCBI GenInfo Identifier:	46909569
NCBI Gene ID:	14463
NCBI Accession:	NP_032118.2
UniProt Secondary Accession:	Q08369,P97491, Q9QZK4, B9EHF7,
UniProt Related Accession:	Q08369
Molecular Weight:	44,580 Da
NCBI Full Name:	transcription factor GATA-4 isoform 2
NCBI Synonym Full Names:	GATA binding protein 4
NCBI Official Symbol:	Gata4
NCBI Official Synonym Symbols:	Gata-4
NCBI Protein Information:	transcription factor GATA-4
UniProt Protein Name:	Transcription factor GATA-4
UniProt Synonym Protein Names:	GATA-binding factor 4
Protein Family:	Transcription factor
UniProt Gene Name:	Gata4
UniProt Entry Name:	GATA4_MOUSE

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.